© The author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Share-Alike License (http://creative-commons.org/licenses/by-sa/4.0/).
Correspondence to: Dr. I. Castro, Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro, 5000-801
Vila Real, Portugal. E-mail: icastro@utad.pt
Uncovering Northeast Portugal grapevine's varietal legacy
D. augusto1), a. a. oliveira2), 3), v. FalCo2), 4) and i. Castro1), 3)
1) Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal 2) Department of Agronomy, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal
3) Centre for the Research and Technology of Agro-Environmental and Biological Sciences, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal
4) Chemistry Research Centre-Vila Real, CQ-VR, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal
Summary
Northeast Portugal comprises the wine denomina-tions "Douro" and "Trás-os-Montes". There are more than one hundred grapevine cultivars registered suita-ble for wine production in these regions (ministerial-or-ders number 1204/2006 and 383/2017), however only a few are actually used for winemaking. In this sense, the identification of the varieties cultivated in past times may widen the number of varieties and can be an im-portant step to take advantage of all the potential of these regions' grapevine biodiversity. The conservation of these vanishing genetic resources boosts a greater products' diversification and it can be considered stra-tegic in the improvement and valorization of "Douro" and "Trás-os-Montes" PDO wines. Moreover, it is im-perative to identify, before to characterize and pre-serve, plants of late-maturing grape varieties planted in regions with extremely high temperatures and precipi-tation deficits which still maintain grape yield and pro-duce very well-known quality wines. Hence, the main goals of this study were to prospect and characterize, through nuclear and chloroplast SSR analysis, plants of old vineyards that constitute a broad representation of the grapevine genetic patrimony of "Douro" and "Trás-os-Montes" regions.
K e y w o r d s : biodiversity; chlorotype; genotyping; mi-crosatellite; Vitis vinifera L.
Introduction
Northeast Portugal comprises the wine denominations "Douro", the oldest demarcated and regulated winemak-ing region in the world, and "Trás-os-Montes" (IVV 2017). "Douro" region represents approximately 22 % of the Por-tuguese vineyard area for wine production, while "Trás-os-Montes" only accounts for 7 % (IVV 2017). Furthermore, these regions characterized by their mountains with steep slopes and valleys provide distinct microclimates and, con-sequently, a high genetic diversity due to the evolutionary need of grapevine adaptation to different climatic condi-tions (graça 2012).
However, this rich heritage in grapevine varieties which contributes so much to an environmentally sustain-able viticulture is at risk, due to the limitation in the use of autochthonous varieties in the different Protected Des-ignations of Origin (PDO) regions and to European Un-ion incentives for vineyards' restructuring and conversUn-ion to commercially available clones (EU 2014). Hence, the grapevine genetic pool is diminishing and crop vulnerabil-ity to abiotic and biotic stresses is increasing.
Several studies are reporting on the genetic diversity among Portuguese grape varieties based on nuclear and/ or chloroplastidial microsatellites (lopes et al. 1999 and
2006, Baleiras-Couto and Eiras-Dias 2006, AlmaDanim
et al. 2007, Cunha et al. 2009, Veloso et al., 2010, Castro
et al. 2011 and 2013, Ferreira et al. 2015). Nonetheless,
since the consumers are increasingly requiring high-quality and diversified wines, a continuous grape varietal prospec-tion is demanded.
Other studies have already revealed the impact of cli-mate change in the advance, i.e. earlier timing, of grape-vine phenological stages (Fraga et al. 2016a and b, Jones
and alves 2012). The maturation of berries is likely to
take place earlier under warmer conditions posing a major challenge to the Portuguese wine-making sector (Fraga
et al. 2016a, Jones and alves 2012). A possible response
towards this projected future warming in vineyards is to preserve late-maturing grape varieties in order to cope with the extreme hot temperatures and precipitation deficits reg-istered in our country.
Hence, the main goal of this study was the molecular identification in old vineyards of a broad representation of grapevine cultivars patrimony of "Douro" and "Trás-os-Montes" regions contributing to deepen the knowledge of Northeast Portugal grapevine's genepool.
Material and Methods
S a m p l i n g a n d D N A e x t r a c t i o n : To ana-lyse ancient genetic diversity of V. vinifera in "Douro" and "Trás-os-Montes" PDO regions, 165 plants were sampled across six different old mixed variety vineyards, all pre-dating the 1970s (Fig. 1; Tabs 1, 2 and suppl. Tab. S1). The selection of plants was based on the difficulty of their
morphological identification by ampelographers. All plants were labelled in the vineyards and young leaves collect-ed in "Trás-os-Montes" (year 2017) and in "Douro" (year 2018) sampling locations and stored at -80 °C. Genomic DNA was extracted according to Doyle and Doyle (1990), with some modifications. Total purified DNA was detected by 1.0 % (w/v) agarose gel electrophoresis containing Gel-Green™ Nucleic Acid Gel Stain 1x (Biotium) and stored at -20 °C until use.
S S R a m p l i f i c a t i o n a n d g e n o t y p i n g : In this study, a set of 6 nuclear microsatellite (nSSR) loci was used - VVS2, VVMD5, VVMD7, VVMD27, ssrVrZAG62 and ssrVrZAG79 (OIV 2009). The forward primer of each pair was fluorescently labelled with 6-FAM (VVMD5 and VVMD27), TET (VVMD7 and ssrVrZAG62) or HEX (VVS2 and ssrVrZAG79). Two multiplex PCRs were car-ried out as previously described by Castro et al. (2011), with 0.75 μM BSA added to each 20-μL reaction mixture. The three most polymorphic chloroplastidial microsatel-lite (cpSSR) loci in grapevine (Arroyo-garCía et al. 2002, Cunha et al. 2009) - ccmp3, ccmp5 and ccmp10 - were also amplified for all distinct grapevine varieties, according to Castro et al. (2013). The forward primer of each pair was fluorescently labelled with 6-FAM (ccmp3), VIC (ccmp5) or NED (ccmp10). Fluorescently labelled cp and nSSR products were separated by capillary electrophoresis using the ABI PRISM® 3730 automated sequencer (Applied Bi-osystems, Life Technologies) and GeneScanTM 500 LIZ® (Applied Biosystems, Life Technologies) as the internal
lane size standard. Data produced were analyzed by Peak Scanner v1.0 software (Applied Biosystems). The sizes of the amplicons were scored in base pairs (bp) based on the relative migration of the internal size standard. The nSSR profiles obtained were compared to those of the Vitis Inter-national Variety Catalogue (VIVC) database (http://www. vivc.de).
Analysis of nuclear SSR data: To establish the genetic relationships among V. vinifera cultivars, allelic data were directly used to generate a squared distances matrix using NTEdit software. The similarity matrix was processed by NTSYS-pc software (Version 2.20; RohlF 2005) based on
the coefficient DICE and UPGMA method was applied to obtain a dendrogram.
Results and Discussion
Vitis vinifera L. varieties identification for diversity
as-sessment: The prospection included a total of 165 samples from "Douro" and "Trás-os-Montes" PDO regions, 37 of which, accounting for 13 genotypes, could not be identified by comparison with the VIVC database and are being fur-ther studied (Tab. 2). This work focuses on the remaining 128 samples, which were identified through comparison of their genetic profiles generated via nuclear SSR loci ampli-fication. In total, 34 distinct grapevine varieties were tected (Tabs 1 and suppl. Tab. S2), from which 22 were de-scribed as autochthonous to Portugal (Fig. 2; AlmaDanim et Fig. 1: Geographical location of the 165 samples in the six
loca-tions of "Douro" and "Trás-os-Montes" PDO regions and the age of vineyards (adapted from IVV 2017).
Fig. 2: Distribution of grapevine varieties in PDO "Douro" and PDO "Trás-os-Montes" according to their vineyard's total area (IVV 2017). Thirty-four genotypes were identified, belonging to either the most cultivated varieties in Portugal (which means a representativity superior to 1 % of total area) or to the minority varieties' group. Foreign grape cultivars were also detected, and the ones identified with an asterisk are cultivated in the Iberian Peninsula.
Ta b l e 1
List of the 34 Vitis vinifera L. varieties identified and respective samples code, synonymies, berry colour and chlorotype
Sample code1 Cultivar prime name variety VIVC
number
Synonymies in
Portugal berry skinColour of 2 haplotypecpSSR 3 References4
QSI19 Alvarelhão-Ceitão (D)* 368 R A c
Vs25, Vs26, QSI9, QSI14, QSI32, Sd1,
Sd16, Sd59, Sd60, Qs7 Trousseau (D,T) 12668 Bastardo N A a,g
Qs9 Baga 885 N A b
QSI8 Black Monukka 17452 N C d
V28 Camarate-Tinto* 2018 N A a
Sd2, Sd3, Sd4, Qs14 Casculho (D)* 14149 N A this study
V33 Castelão (D,T)* 2324 N A a
QSI2 Chasselas (D) 2473 B D b
Sd20 Cornifesto (D,T)* 2846 N A a
QSI7, QSI22 Dodrelyabi 3616 N B d
Vs21 Donzelinho-Roxo* 17677 R D this study
Ag6 Folha-de-Figueira (D)* 14142 Dona-Branca B A d
Ag2, Ag12, Sd47, Sd63 Gouveio (D,T)* 12953 B A a,b
Qs18 Grand Noir (D) 5012 N A d
QSI3, QSI10 Hebén (D) 5335 Mourisco-Branco B A d
Vs23 Jerónimo de Tudela 5692 N A d
Vs2, Vs4 Mantúo (D) 2520 Diagalves B A a,c
Ag11, Vs12, Vs14, Vs15, Vs19, Vs20,
QSI16, Sd58, V20, Qs13 Marufo (D,T)* 8086 N D a,b
Ag1 Molar* 15678 Tinta-Negra N A g
Ag7, Vs5, Vs7, Vs10, Sd15, Sd17, Sd18, Sd28, Sd36, Sd38, Sd39, Sd41, Sd42,
Sd48, Sd54, Sd55, Qs10 Mouratón (T) 8082 Tinta-Gorda N A d
QSI24, QSI28 Palomino Fino (D) 8888 Malvasia-Rei B D a
Sd25, Sd27 Perlette 9168 B A d
Qs19 Rufete (D,T)* 10331 N A a
Vs9 Samarrinho (D,T)* 15684 Budelho B D a
Vs11, Vs18, QSI6, QSI18, QSI20, QSI23, QSI29, QSI33, QSI34, Sd5, Sd10, Sd19,
Sd22, Sd29, Sd34, Sd37, Sd61 Síria (D,T)* 2742 B A a
Vs24, Sd44, Sd45, V4, V6, V9, V14, V15,
V17 Tempranillo (D,T) 12350 Aragonez N A a,b
Sd57 Tinta-Barroca (D,T)* 12462 N D b,f Vs3, Vs8, V34 Tinta-Carvalha (D,T)* 12467 N D b V12, V19, Qs15 Tinta-Francisca (D)* 15686 N A b Vs16, Sd50 Tinto-Cão (D,T)* 12500 N A b,f Qs6 Touriga-Nacional (D,T)* 12594 N A b Sd12 Touriga-Franca (D,T)* 12593 N D a,b,f
Vs13, Vs27, QSI4, QSI17, QSI21, QSI25, QSI26, QSI27, QSI30, QSI31, QSI35, Sd8, Sd11, Sd51, Sd62, V5, V22, V27, V30, V40, V46
Trincadeira (D,T)* 15685 N D a,b
Qs12 Vinhão (D,T)* 13100 Sousão N A b
1 Ag - Aguieiras; Vs - Vassal; QSI - Quinta de Santa Isabel; Sd - Sendim; V - Quinta dos Lagares; Qs - Quinta do Seixo. 2 N - Noir, R - Rouge, B - Blanc. 3 Designation according to Arroyo-garCía et al., 2002.
4 a - Cunha et al., 2009; b - Castro et al., 2013; c - Imazio et al., 2006; d - VIVC.de; e - Ferreira et al., 2015; f - Baleiras-Couto and Eiras-Dias, 2006; g - moita maçanita et al., 2018. Varieties presumably autochthonous to Portugal were marked by asterisk and those
authorized in 'Douro' and/or 'Trás-os-Montes' PDO regions with (D) and/or (T), respectively.
al. 2007, Veloso et al. 2010, Castro et al. 2011). Genetic relations among these grape varieties are shown in the sup-pl. Fig. S1. According to VIVC data, the Portuguese culti-vars 'Mourisco-Branco', 'Tinta Gorda', 'Diagalves', 'Arag-onez' and 'Malvasia-Rei' are synonymies of the Spanish cultivars 'Hében', 'Mouratón', 'Mantúo', 'Tempranillo' and 'Palomino Fino'. Twenty-seven of the 34 varieties are au-thorized for red and white wine production under "Douro"
and "Trás-os-Montes" Protected Denomination of Origin (IVV 2017). Not on the lists of grape varieties suitable for the production of wine in these PDO are the following va-rieties: 'Black Monukka', 'Camarate-Tinto', 'Dodrelyabi', 'Donzelinho-Roxo', 'Jerónimo-de-Tudela', 'Molar' and 'Per-lette'. Additionally, 12 out of the 34 varieties identified are not protected under the clonal selection programme ('Alva-relhão-Ceitão', 'Diagalves', 'Dozelinho-Roxo', 'Casculho',
'Cornifesto', 'Folha-de-Figueira', 'Malvasia-Rei', 'Marufo', 'Mourisco Branco', 'Samarrinho', 'Tinta Carvalha' and 'Tin-ta Gorda'). Another fact to 'Tin-take into account was that seven red varieties ('Cornifesto', 'Marufo', 'Rufete', 'Tinta-Bar-roca', 'Tinta-Francisca', 'Tinto-Cão' and 'Vinhão') and five white ('Diagalves', 'Gouveio', 'Malvasia-Rei', 'Samarrinho' and 'Síria') listed on these PDO regions are described as late-maturing ones (Böhm 2007, CarDoso 1995). Several studies have already alerted to the advance of grapevine phenological stages due to climate change, which can lead to a significant impact on grape yield and wine quality in Portugal (Jones and alves 2012, Fraga et al. 2016a). Hence, high quality PDO wines from late-maturing grape cultivars will likely need to be considered under a future warmer climate.
Vitis vinifera L. chlorotypes and cpSSR
polymor-phisms: In the three chloroplastidial SSR loci amplified for the 34 grapevine varieties genotyped in this study, at least 2 allele variants were detected for each ccmp locus (suppl. Tab. S3). The combination of these alleles provided four different chlorotypes which were designated accord-ing to arroyo-garCía et al. (2002) (see Tab. 1 and suppl. Tab S3). Chloroplastidial haplotype A was the most fre-quent, and it was observed in 67.6 % of the grape culti-vars, followed by chlorotype D with 26.5 %. Haplotype A characterizes the Iberian Peninsula cultivars, which is referred as a secondary centre of domestication of
Vi-tis vinifera L. ssp. vinifera, whereas haplotype D is more
commonly observed in eastern European grape varieties (arroyo-garCía et al. 2006, Cunha et al. 2009, Castro
et al. 2013, moita maçanita et al. 2018). Chlorotypes B and C were only present in the foreign 'Dodrelyabi' and 'Black Monukka' cultivars, respectively. Chlorotypes were for the first time determined for the Portuguese 'Casculho' and 'Donzelinho-Roxo' grape varieties, as no previous ref-erences were found in the literature; they revealed to be
haplotype A based on the 3 polymorphic ccpm loci ana-lysed. 'Casculho' chlorotype was consistent with its pro-genitors (also A haplotypes), the Portuguese 'Alfrocheiro' and the Iberian 'Cayetana Blanca' varieties (ZinelaBiDine et
al. 2012, Cunha et al. 2013). Also, 'Casculho' is a sibling of 'Cornifesto' and both share approximately 86 % of similar-ity based on 6 nSSR loci (ZinelaBiDine et al. 2012, suppl. Fig. S1). However, no information was found regarding 'Donzelinho-Roxo' origin.
Conclusions
This work contributed to the identification of 128 plants in the PDO's "Douro" and "Trás-os-Montes" which were grouped into 34 different genotypes/varieties. Four chlorotypes (A, B, C and D) were also detected and helped to infer about the genetic origin of these 34 autoch-thonous and foreign grapevine varieties. The microsatellite analysis was very useful in the identification and discrimi-nation of plants analysed and overcomes the ampelography difficulties in grapevine prospections in order to contribute to the ultimate goal of conserving grape's varietal legacy in Northeast Portugal. Further morphological, agronomical and oenological analysis are being undertaken to comple-ment the molecular data presented in this study.
This study is the base to preserve the intravarietal ge-netic variability that must exist in the plants of known vari-eties identified within old vineyards. Moreover, thirty-five percent of the varieties identified are not protected under the national clonal selection programme and, in this sense, plant material of these genotypes must be propagated at different viticulture stations, particularly at the Portuguese Association for the Diversity of Grapevine. Viticulture fac-es new challengfac-es to rfac-espond to consumer's demands and particularly to climate change. Thus, characterization and Ta b l e 2
Nuclear SSR profiles of 13 new genotypes
New
genotypes Sample code1
SSR sizes (bp) VVS2 VVMD5 VVMD7 VVMD27 VrZAG62 VrZAG79 1 Ag3, Ag 5, Vs6 136 150 230 234 241 243 177 185 189 191 245 249 2 Ag8 136 158 222 232 239 239 177 185 189 189 245 249 3 Ag9 144 152 224 234 239 239 185 185 191 191 243 255 4 Ag10, Sd6, Sd7, Sd14, Sd21,Sd23, Sd31, Sd32, Sd33, Sd56 136 150 230 232 243 257 177 185 191 197 243 245 5 Ag11 136 150 230 232 243 247 177 185 191 197 243 257 6 Ag12 142 158 222 224 241 243 179 185 191 195 245 249 7 Vs17 140 152 230 236 251 253 177 177 191 191 245 255 8 Vs22 142 152 224 234 241 243 185 185 189 195 249 255
9 QSI1, QSI12, QSI13, QSI15 132 144 224 236 241 249 181 191 191 197 249 255
10 QSI5, QSI11 132 134 222 232 247 255 175 181 205 205 249 249
11 Sd9, Sd13, Sd24, Sd30, Sd35,Sd43, Sd46, Sd49, Sd52, Sd53 144 150 232 234 249 253 177 185 203 207 245 249
12 Sd40 132 150 232 234 239 239 177 185 189 191 245 249
13 V32 142 144 224 228 239 243 179 179 191 195 245 255
preservation of this grapevine genetic background pros-pected, mainly of late-ripening cultivars, is of crucial im-portance to face alterations in temperature, precipitation, frequency and duration of extreme weather events, and also their resulting abiotic consequences.
Acknowledgements
This work was supported by the project IN-NOVINE&WINE – Vineyard and Wine Innovation Plat-form – NORTE-01-0145-FEDER-000038, co-financed by the European Regional Development Fund (ERDF) through the Norte2020 (Northern Regional Operational Programme 2014/2020) and National Funds by FCT - Por-tuguese Foundation for Science and Technology, under the project UID/AGR/04033/2019. The authors would like to thank to Comissão Vitivinícola Regional de Trás-os-Mon-tes (CVRTM), Sogrape Vinhos and Quinta dos Lagares for kindly allowing the sampling of plant material.
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